Tools and Technologies I
Statement of Benefit to California:
The goal of the proposed study is to develop a method for genetic modification of human embryonic stem cells (hESC) with high precision and efficiency. The approach involves development of a baculoviral vector (BV) system, which can accommodate large DNA inserts, together with specifically engineered zinc-finger nucleases (ZFN) that can stimulate site-specific DNA integrations. In a first aim, conditions will be optimized for using BV-mediated DNA delivery to target the CCR5 locus in hESC. In a second aim, a ZFN capable of targeting the Oct-4 locus will be designed, using a novel display technique. In aim 3, the applicants will use the developed tools to create an Oct4 reporter hESC line. The proposed research could have an important impact on stem cell research. The ability to genetically manipulate hESC by delivering large genomic fragments to specific genetic loci would be a significant advancement that could facilitate marking genes or making precise changes in stem cell genomes. This will aid the study of molecular regulation of stem cell differentiation and facilitate the development of hESC-based disease models. The approach could also enable the generation of genetically corrected patient-specific induced pluripotent (iPS) cells from patients with genetic disorders. Reviewers found the rationale for the proposed study to be solid and compelling. Research plans were appropriately supported by previous work in the investigators’ labs. Reviewers judged the aims as straightforward, proposed experiments well described, and alternative strategies outlined clearly. Very convincing preliminary data supported feasibility of the approach. Although the project was viewed as ambitious and somewhat risky, reviewers felt that the potential benefits as well as the solid investigative plan portend good prospects for success. One minor criticism related to the choice of Oct4 as a reporter locus, as such a reporter hESC line already exists. Furthermore, an attempt should have been made to explore the strength of the BV delivery system, its capacity to deliver large DNA fragments. The applicant is a productive scientist with expertise in viral vector technology and polymer chemistry, and the co-investigator brings key expertise with the novel display technology to the project. The assembled research team appears substantial and well qualified to carry out the proposed investigation. Overall, this is a well-designed proposal for creating a valuable new tool for stem cell research. Reviewers were enthusiastic about the approach and its likelihood for success.